Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity.

Autor: Sinijarv H; Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia., Wu S; Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia., Ivan T; Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia., Laasfeld T; Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia. Electronic address: tonis.laasfeld@gmail.com., Viht K; Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia., Uri A; Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia. Electronic address: asko.uri@ut.ee.
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2017 Aug 15; Vol. 531, pp. 67-77. Date of Electronic Publication: 2017 May 18.
DOI: 10.1016/j.ab.2017.05.017
Abstrakt: High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest K d value that can be accurately determined with the method is 60 fM. We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of K d determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.
(Copyright © 2017 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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